Vac14 controls PtdIns(3,5)P₂ synthesis and Fab1-dependent protein trafficking to the multivesicular body

Background: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P-2, a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. Results: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P-2, and hyperosmotic shock does not stimulate PtdIns(3,5)P-2 synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. Conclusions: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P-2 synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P-2, for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.